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J Biol Chem, Vol. 275, Issue 20, 14799-14808, May 19, 2000
From the Vanillyl-alcohol oxidase is a flavoprotein
containing a covalent flavin that catalyzes the oxidation of
4-(methoxymethyl)phenol to 4-hydroxybenzaldehyde. The reaction proceeds
through the formation of a p-quinone methide intermediate,
after which, water addition takes place. Asp-170, located near the
N5-atom of the flavin, has been proposed to act as an active site base.
To test this hypothesis, we have addressed the properties of D170E,
D170S, D170A, and D170N variants. Spectral and fluorescence analysis, together with the crystal structure of D170S, suggests that the Asp-170
replacements do not induce major structural changes. However, in D170A
and D170N, 50 and 100%, respectively, of the flavin is non-covalently
bound. Kinetic characterization of the vanillyl-alcohol oxidase
variants revealed that Asp-170 is required for catalysis. D170E is
50-fold less active, and the other Asp-170 variants are about
103-fold less active than wild type enzyme. Impaired
catalysis of the Asp-170 variants is caused by slow flavin reduction.
Furthermore, the mutant proteins have lost the capability of forming a
stable complex between reduced enzyme and the p-quinone
methide intermediate. The redox midpoint potentials in D170E (+6 mV)
and D170S (-91 mV) are considerably decreased compared with wild type
vanillyl-alcohol oxidase (+55 mV). This supports the idea that Asp-170
interacts with the protonated N5-atom of the reduced cofactor, thus
increasing the FAD redox potential. Taken together, we conclude that
Asp-170 is involved in the process of autocatalytic flavinylation and is crucial for efficient redox catalysis.
The atomic coordinates and the structure factors (code 1DZN) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
Asp-170 Is Crucial for the Redox Properties of
Vanillyl-alcohol Oxidase*
§,
,**
Department of Biomolecular Sciences,
Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands and the ¶ Department of
Genetics and Microbiology, University of Pavia,
Via Abbiategrasso 207, 27100 Pavia, Italy
*
This work was performed within the framework of the
Innovation Oriented Research Program Catalysis of the Dutch Ministry of Economy Affairs (Project IKA 96005). This work was supported by the
Ministero dell'Universita e Ricerca Scientifica e Tecnologica (Project
Biosintesi del NAD: Studi Biochimici, Biologia, Strutturale e Sviluppo
Razionale di Farmaci) and the Consiglio Nazionale delle Ricerche
(Target Project on Biotechnology).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Supported by a long term fellowship from the European
Molecular Biology Organization.
**
To whom correspondence should be addressed. Tel.: 31-317-482861;
Fax: 31-317-484801; E-mail: willem.vanberkel@fad.bc.wau.nl.
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